The peptide substance P (SP) is an eleven amino acid peptide (FIG. 1, SEQ ID NO:6) that belongs to the tachykinin family of neuropeptides. Known mammalian tachykinins (neurokinins) include neurokinin A, neurokinin B, neuropeptide K, and neuropeptide g. All tachykinins share the carboxy-terminal sequence Phe-Xaa-Gly-Leu-Met-NH.sub.2 (where Xaa is an aromatic or aliphatic amino acid) (SEQ ID NO:61). The mammalian tachykinins are produced by neurons in the central and peripheral nervous system where they are predominantly localized in the nerve terminals (Escher, E. and Regoli, D. (1989) in Peptide Hormones as Prohormones: Processing, Biological Activity, Pharmacology (Martinez, J., ed.) pp 26-52, Ellis Horwood Limited, West Sussex, England).
Neurotransmitter and neuromodulator functions of SP include peripheral vasodilation, smooth muscle contraction, pain transmission (nociception), stimulation of exocrine secretions, and immunomodulation (for a review see Escher, E. and Regoli, D. (1989) in Peptide Hormones as Prohormones: Processing, Biological Activity, Pharmacology. (Martinez, J., ed.) pp 26-52, Ellis Horwood Limited, West Sussex, England). There is also evidence that SP has memory-modulating and reinforcing effects. Huston et al. (1993) Psychopharmacology 112: 147-162 have suggested a possible link between SP and the impairment in associative functioning accompanying Alzheimer's disease.
The pharmacological importance of substance P is further indicated by recent studies that suggest that SP has a role in angiogenesis (e.g., Fan, T. et al. (1993) Brit. J. Pharmacol. 110: 43-49). Fan et al. suggest that the positive interaction between SP and the cytokine interleukin-1 alpha (IL-1a) may be important in the angiogenic cascade leading to a variety of diseases characterized by excessive neovascularization (e.g., rheumatoid arthritis, atherosclerosis, diabetic retinopathy and cancer). Blocking substance P activity, therefore, may effectively reduce the progression of the disease.
A method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, entitled Systematic Evolution of Ligands by Exponential Enrichment, now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled Nucleic Acid Ligands, now issued as U.S. Pat. No. 5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled Nucleic Acid Ligands, now U.S. Pat. No. 5,270,163 (see also PCT/US91/04078), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
The basic SELEX method may be modified to achieve specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled Method for Selecting Nucleic Acids on the Basis of Structure, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled Photoselection of Nucleic Acid Ligands describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed "counter-SELEX." U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Specific SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled High Affinity Nucleic Acid Ligands Containing Modified Nucleotides, that describes oligonucleotides containing nuclgotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines, as well as specific RNA ligands to thrombin containing 2'-amino modifications. U.S. patent Application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). Each of these applications is specifically incorporated herein by reference.
The development of high affinity ligands of SP are useful as diagnostic and pharmacological agents. Specifically, ligands capable of inhibiting SP would be useful in the treatment or monitoring treatment (i.e., diagnostic applications) of numerous diseases, including angiogenic diseases such as rheumatoid arthritis, atherosclerosis, diabetic retinopathy, and cancer. Herein described are high affinity nucleic acid ligands of SP. Considering the size of SP, it can be assumed that ligands of SP will be inhibitors of SP.